Sherief et al.

BMC Pediatrics (2017) 17:69

DOI 10.1186/s12887-017-0820-1

RESEARCH ARTICLE Open Access

Premature atherosclerosis in children with

beta-thalassemia major: New diagnostic
marker
Laila M. Sherief1*, Osama Dawood2, Adel Ali3, Hanan S. Sherbiny3, Naglaa M. Kamal4, Mohamed Elshanshory5,
Osama Abd Alazez6, Mohamed Abd Alhady3, Mohamed Nour3 and Wesam A. Mokhtar3

Abstract
Background: Early vascular alteration, atherosclerosis and coronary artery disease have emerged as important
cardiovascular complications among beta-thalassemia major (B-TM) patients. The aims of the current study were to
assess the prevalence of premature atherosclerosis among our B-TM patients, and to investigate the diagnostic
value of serum Osteoprotegerin assay as an early biomarker for atherosclerosis.
Methods: This cross-sectional study was conducted at Hematology unit - Pediatric Department, Zagazig University
Children Hospital- Egypt in the period from March 2014 to March 2015. A total of 115 children were enrolled in the
current study; as sixty-five (65) children with beta thalassemia major aged 5–18 years, on regular blood transfusion
regimen represented the patient group. While fifty (50) healthy children, with comparable age and gender, were
assigned as control group. All participants were subjected to history taking, thorough clinical examination and
laboratory investigations including; complete blood count, liver and kidney function tests, C- reactive protein, lipid
profile, serum ferritin and serum Osteoprotegerin (OPG) assay. Also, carotid artery intima media thickness (CAIMT)
was performed by duplex ultrasound for patients and controls.
Results: Our B-TM patients were transfusion-dependent for as long as 8.5 ± 3.8 years with significantly higher serum
ferritin levels (2490 ± 1579 ng/dl vs 83 ± 32 ng/dl, p = 0.001), C-reactive protein (5.7 ± 5.7 vs 0.9 ± 0.9), liver enzymes
and bilirubin when compared to controls. Significantly higher serum triglyceride (128 ± 20 vs 101 ± 7 mg/dL, p = 0.
009) and atherogenic index of plasma (0.45 ± 0.12 vs 0.22 ± 0.04, p = 0.001) were recorded in patients than
comparisons. On the contrary, total serum cholesterol (116 ± 16 vs 143 ± 5, p < 0.001), low density lipoprotein-
cholesterol (LDL-C) (44 ± 9 vs 73 ± 6, p < 0.001) and high density lipoprotein cholesterol (HDL-C) (39 ± 2 vs 61 ± 5,
p < 0.001), were significantly lowered in patients versus normal peers. Carotid arteries intima media thickness (CAIMT)
of both side were significantly increased for patients (Rt 0.62 ± 0.2 vs. 0.29 ± 0.07 mm, p = 0.001 & Lt 0.66 ± 0.17 vs 0.29
± 0.05 mm, p = 0.001) when compared with healthy controls, and showed positive correlation with, serum triglyceride,
atherogenic index of plasma, and serum Osteoprotegerin levels. ELISA assay of serum Osteoprotegerin (OPG) revealed
significantly higher levels for thalassemia patients than matched healthy controls (427 ± 102 vs. 324 ± 126 pg/ml, p = 0.
02). Of particular interest is the obvious positive correlation between OPG levels and CAIMT of both sides
(Rt r 0.54, p = 0.001 &Lt r 0.479, p = 0.001) and also with serum triglycerides (r 0.374, p = 0.03).
(Continued on next page)

* Correspondence: Lamesh25@yahoo.com
1
Pediatrics and Pediatric Hematology and Oncology, Faculty of Medicine,
Zagazig University, Zagazig, Egypt
Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Sherief et al. BMC Pediatrics (2017) 17:69
(Continued from previous page)
Conclusions: Subclinical atherosclerosis started prematurely in children with beta- thalassemia. Carotid artery intima
media thickness represented a simple, accurate and non-invasivemodality for early detection ofatherosclerosis.
It was correlated well with serum Osteoprotegerin; this finding highlighted the possible validity of OPG assay as an
early predictor of atherosclerosis in thalassemia children.
Keywords: Beta-thalassemia, Carotid artery intima media thickness, Osteoprotegerin, premature atherosclerosis
Background
Beta-thalassemia represents the commonest cause of
hemolytic anemia in Egypt with carrier rate ranges from 9–
10% [1]. Beta- thalassemia major (B-TM) patients usually
present within the first two years of life with severe anemia
requiring regular red blood cell transfusions [2]. Early vas-
cular alteration, atherosclerosis and coronary artery diseases
have emerged as important cardiovascular complications
among B-TM patients [3].
Atherosclerosis is a slow progressive disease that may
start at childhood [4]. In atherogensis, arterial wall morpho-
logical changes occur during a presumably long subclinical
lag phase, and characterized by gradual thickening of the
intima [5]. Beside the traditional diagnostic methods such
as angiography and stress-testing [6], measurement of the
intima-media thickness of the large arteries, especially the
carotids, has emerged as one of the method of choice for
determining the anatomical extent of arterial wall deterior-
ation and for assessing cardiovascular risk [5]. Several
investigators have recommended the clinical use of this
technique for detecting subclinical (asymptomatic) athero-
sclerosis and for identifying subjects at high-risk [7–9].
Circulating markers of atherosclerosis are associated with
increased vascular risk; one of the new biomarkers of ath-
erosclerosis is Osteoprotegerin (OPG) [10]. Osteoproteg-
erin is a cytokine of the tumor necrosis factor (TNF)
receptor superfamily and is classed as an osteoclastogenesis
inhibiting factor [11]. In the endothelial cell, OPG is associ-
ated with Von-Willebrand factor within secretory granules
called Weibel-Palade bodies. Upon stimulation with TNF
or interleukin-1 in vitro, the OPG- Von-Willebrand factor
complex is secreted in the surrounding growth medium.
This endothelial activation by pro-inflammatory cytokines
is one of the possible sources of circulating OPG in patients
with active atherosclerosis [12]. Moreover, in view of the
role of vascular smooth muscles in atherosclerosis and
intimal calcification, it is likely that, they are the main
source of increased circulating OPG noted in cardiovascu-
lar disease [13]. Data suggest that OPG is induced by
atherosclerosis and may be upregulated as an incomplete
compensatory response to the vessel insult, possibly thereby
limiting vascular calcification [14]. Osteoprotegerin seems
to prevent arterial calcification but is not able to reverse
calcification once it has occurred [15].
Page 2 of 8
As the maximal potential for prevention and revers-
ibility of atherosclerosis would be expected by interven-
tion at early subclinical stage of the disease, its early
diagnosis in high-risk individuals should be a research
priority. The aims of the current work are; to assess the
frequency of premature subclinical atherosclerosis in
Egyptian B-TM patients by determining their carotid
artery intima-media thickness, to figure-out the associated
clinical and laboratory risk factors, and to evaluate the
validity of OPG assay as a new biomarker for early diagno-
sis of atherosclerosis in these high-risk population.
Methods
Patients
We carried out this cross-sectional, case–control study on
sixty –five (65) Beta-thalassemia major patients aged 5–18
years old. They were on regular follow-up at the Hematology
Unit of Pediatric Department, Zagazig University Children
Hospital- Egypt during the period from March 2014-March
2015. Fifty (50) apparently healthy children with comparable
age and gender distribution were enrolled as controls.
Thalassemia patients enrolled in the current work
were on regular red cell transfusion regimen, 33 cases
were transfused every 4 weeks, while 22 and 10 cases
were transfused every 3 weeks and 2 weeks respectively
to keep their target haemoglobin [16].
All subjects in our study were questioned for known risk
factors for atherosclerosis, patients with history of smok-
ing, hepatic, renal or cardiac diseases were excluded. Also
those with diabetes mellitus or other endocrinopathies,
other hemoglobinopathies, familial hypercholesterolemia
or premature atherosclerosis in their families were also
excluded.
The study protocol was approved by the research and
ethical committee of Faculty of Medicine, Zagazig
University and written informed consent was taken from
parents or guardian of each participant.
Methods
All eligible children (patients and controls) were
subjected to; history taking with special emphasis on
demographic characteristics, disease duration, frequency of
transfusions, iron chelation regimen including type, dose,
duration and compliance. Thorough clinical examination
Sherief et al. BMC Pediatrics (2017) 17:69 Page 3 of 8

including anthropometric measures and all system 4 weeks transfusion regimen, 34% were on 3 weekly regi-
evaluation were performed. Routine laboratory investiga- men while for the remaining 15.3% cases every two weeks
tions for follow up thalassemia patients that included; transfusion was needed to keep target hemoglobin. Deferi-
complete blood count, liver function tests, renal function prone was used as iron chelation in 49% of them, 45%
tests, serum ferritin, C-reactive proteins and hepatitis used Deferasirox while the remaining 6% were still using
markers were taken. desferrioxamine as iron chelation therapy.
Specific tests for evaluation of atherosclerosis were Results of B-TM patients’ clinical evaluations, routine
performed and included 12-h fasting lipid profile, serum and specific laboratory investigations were compared with
Osteoprotegerin (OPG) assay, and carotid artery intima- those of fifty, age and gender, matched healthy children.
media thickness. All blood sampling for patients were Apart from height, no significant difference could be
performed at the day before red blood cell transfusion. detected atdemographic or anthropometric measures be-
Lipid profile was taken after at least 12 h overnight tween patients and controls as shown in Table 1. On the
fasting, it was performed by endpoint method (colorimet- contrary, significant differences were documented when
ric method) and included; total serum cholesterol, high results of complete blood count and routine chemical ana-
density lipoprotein- cholesterol (HDL-C), serum triglycer- lysis were evaluated as displayed in Table 2. B-TM patients
ide (TG). Low density lipoprotein –cholesterol (LDL-C) had significantly higher white blood cell count and plate-
was estimated using Friedewald and Levyformula [17] by lets but much lower hemoglobin concentration than
calculation of (TG)-(TG/5)-(HDL-C). Atherogenic index healthy comparisons. Almost thirty fold rise in serum
of plasma (AIP) is the ratio calculated as log (TG/HDL- ferritin was reported (2490 ± 1579 ng/dl vs 83 ± 32 ng/dl,
C). Serum samples for assay of OPG were separated and p = 0.001) for cases than controls. Significant rise in
stored at −20 C0. It was performed using Human OPG hepatic enzymes, total bilirubin and C-reactive protein
ELISA Kit, Boster Biological Technology Co., Ltd. USA. were also revealed in patients group than healthy peers.
Carotid artery intima-media thickness (CAIMT) mea- Significantly higher serum triglyceride (128 ± 20 vs 101 ±
surements were performed for all participants by the same 7 mg/dl, p = 0.009) and calculated atherogenic index of
experienced vascular radiologist who was blinded to the plasma (0.45 ± 0.12 vs 0.22 ± 0.04, p = 0.001) were recorded
clinical and laboratory details of the examined children. in patients than comparisons. On the contrary, total serum
Duplex ultrasound B-mode and color-coded duplex son-
ography were performed using a (GE LOGIC P5) ultra-
Table 1 Demographic data and Anthropometric measures of
sound system with a 12.0 MHz linear array transducer.
the studied groups
Character Patients Controls Test p-value
Statistical analysis (N = 65) (N = 50)
All data were analyzed using SPSS 22.0 for windows (SPSS Age (years)
Inc., Chicago, IL USA) and MedCal 13 for windows
Mean ± SD 9.5 ± 3.7 10.4 ± 3.7 MW = −0.348 0.729
(MedCal software bvba). Continuous variables were
expressed as mean ± SD while categorical variables were Range 5 – 18 5 – 18
expressed as number (percentages). Continuous variables Sex No (%)
were checked for normality using Shapiro-Wilk test. Inde- Male 37 (57%) 34 (68%) χ2 = 1.048 0.306
pendent student-t test was used to compare the normally Female 28 (43%) 16 (32%)
distributed variables while Mann–Whitney U (MW) test Residence No (%)
was used to compare non-normally distributed variables
Urban 25 (38.5%) 23 (46%) χ2 = 0.062 0.803
between two groups. Categorical variables were compared
by using Chi-square test (X2). Pearson’ correlation coeffi- Rural 40 (61.5%) 27 (54%)
cient and Spearman’ rank correlation were used to assess Weight (Kg)
relationship between normally distributed and non- Mean ± SD 27.5 ± 8.1 33.8 ± 10.8 t = −1.76 0.085
normally distributed variables respectively. All tests are two Range 14 – 45 19 – 60
sided, and p < 0.05 was considered statistically significant. Height (cm)
Mean ± SD 114.7 ± 21.8 132 ± 22 t = −2.15 0.038
Results
Sixty-five B-thalassemia major (B-TM) patients were re- Range 95 – 155 100 – 168
cruited in the current work, their mean age was 9.5 ± BMI (Kg/m2)
3.7 years (ranged 5–18 years), and 57% of them were Mean ± SD 18.1 ± 2.2 21.5 ± 6.9 MW = 1.45 0.191
males. All of them were transfusion-dependent with ill- Range 16 – 21 16 – 33
ness duration ranged from 4–17 years (mean 8.5 ± MW Mann Whitney U test, χ2 Chi-square test, p < 0.05 is significant,
3.8 years), almost half of them (50.7%) were on every t independent Student t-test, BMI Body mass index
Sherief et al. BMC Pediatrics (2017) 17:69 Page 4 of 8

Table 2 Hematological and biochemical parameters of the ELISA assay of serum Osteoprotegerin (OPG) revealed
studied groups significantly higher levels for thalassemia patients than
Parameter Patients Controls Test p-value matched healthy controls (427 ± 102 vs 324 ± 126 pg/ml,
(N = 65) (N = 50) p = 0.02) as expressed in Fig. 2. Of particular interest is
WBC (×103/mm3) the obvious positive correlation between OPG levels and
Mean ± SD 11.3 ± 4.6 7.3 ± 1.8 MW = 2.5 0.013 CAIMT of both sides (Rt r 0.549, p = 0.001 &Lt r 0.479,
Range 9 – 18.3 5–9 p = 0.001) and also with serum triglycerides (r 0.374, p =
Hb (gm/dl)
0.03) as shown among other parameter in Table 3.
Duplex ultrasonographic Carotid arteries intima media
Mean ± SD 6.8 ± 0.9 12.6 ± 0.7 t = −17.5 <0.001
thickness (CAIMT) of both side were significantly increased
Range 5 – 8.3 11.8 –14.2 for patients (Rt 0.62 ± 0.2 vs. 0.29 ± 0.07 mm, p = 0.001 & Lt
PLT (×103/mm3) 0.66 ± 0.17 vs 0.29 ± 0.05 mm, p = 0.001) when compared
Mean ± SD 478.4 ± 279.4 276 ± 77.6 MW = 2.2 0.030 with healthy controls as described in Table 4. These
Range 136 – 1428 170 – 405 findings have pointed to the high frequency of
SGOT (IU/L)
atherosclerosis among thalassemia group. Documented
positive correlation of CAIMT with serum triglyceride
Mean ± SD 52.3 ± 39.4 30.4 ± 6.8 MW = 2.74 0.020
andatherogenic index of plasma have supported the
Range 9 – 160 20 – 40 previous data. Significant positive relationship between
SGPT (IU/L) CAIMT and S. Osteoprotegerin (OPG) was also ascer-
Mean ± SD 46.4 ± 38.4 29.7 ± 6.8 MW = 2.36 0.020 tained in our thalassemia patients as shown in Table 5.
Range 8 – 155 19 – 38
Total bilirubin (mg/dl)
Discussion
In the current study we tested the hypothesis that
Mean ± SD 1.4 ± 0.6 0.6 ± 0.2 MW = 3.84 0.001
chronic hemolytic anemia may lead to vascular damage
Range 0.6 – 2.8 0.4 – 0.9 and premature atherosclerosis in B-TM patients. Our re-
Direct bilirubin sults documented significantly higher carotid artery
Mean ± SD 0.2 ± 0.1 0.2 ± 0.1 MW = −0.147 0.885 intima-media thickness (CAIMT) of both sides among
Range 0.1 – 0.5 0.1 – 0.4 B-TM patients than matched controls (p < 0.001), a find-
Total Proteins (gm/dl)
ing that provided evidence to the real risk of atheroscler-
osis for these patients.
Mean ± SD 6.1 ± 1.3 6.8 ± 0.8 t = −1.462 0.154
As endothelial dysfunction and increased arterial thick-
Range 4.5 – 8 5.2 – 8 ness are important risk factors for the development of ath-
Urea (mg/dl) erosclerosis [18], several studies have reported the
Mean ± SD 29.6 ± 6.9 27.5 ± 5.6 MW = −0.167 0.847 measurement of arterial intima-media thickness as a good
Range 21 – 41 15 – 37 determinant of subclinical atherosclerosis [19–21]. In-
Creatinine (mg/dl)
creased CAIMT have been described as a mirror of ath-
erosclerotic burden, and a predictor of subsequent events
Mean ± SD 0.5 ± 0.3 0.4 ± 0.2 t = 0.341 0.766
including myocardial infarction and stroke [8, 19]. Because
Range 0.3 – 0.8 0.2 – 0.7 of its quantitative value, it has been used more and more
CRP in clinical trials [8], and noticeably trusted in detecting
Mean ± SD 5.7 ± 5.7 0.9 ± 0.9 MW = 2.6 0.012 pre-clinical (asymptomatic) atherosclerosis in clinical set-
Range 1 – 21 0–2 ting [7]. Depending on the aforementioned advantages of
S. ferritin (ng/dl)
this modality, our team and other researchers [19–21]
have used this technique as a gold standard for early de-
Mean ± SD 2490 ± 1579 83 ± 32 MW = 4.7 <0.001
tection of atherosclerosis. Few studies have been per-
Range 653 – 8406 30 – 127 formed on adults and adolescent patients with B-TM
MW Mann Whitney U test, SGOT Aspartate transaminase, t independent patients and showed significant increase in their CAIMT
Student t-test, SGPT Alanine transaminase, p < 0.05 is significant, CRP
C- reactive protein [21, 22] but to the best of our knowledge this work is the
first to evaluate premature atherosclerosis in Egyptian B-
cholesterol (116 ± 16 vs 143 ± 5, p < 0.001), low density TM children with this modality.
lipoprotein-cholesterol (LDL-C) (44 ± 9 vs 73 ± 6, p < 0.001) There are limited data concerning atherosclerosis risks in
and high density lipoprotein cholesterol (HDL-C) (39 ± 2 vs these patients, we evaluated clinical and laboratory parame-
61 ± 5, p < 0.001), were significantly lowered in patients ver- ters that may be relevant to vascular injury and atherogen-
sus normal controls as displayed in Fig. 1. sis. Clinical characteristics didn’t show any difference from
Sherief et al. BMC Pediatrics (2017) 17:69 Page 5 of 8

Fig. 1 Lipid profile distribution of the studied groups: bar represent mean; Y-error bar represent 95% confidence interval of mean

Fig. 2 Serum Osteoprotegerin (pg/ml) distribution of the studied groups: bar represent mean; Y-error bar represent 95% confidence interval of mean
Sherief et al. BMC Pediatrics (2017) 17:69 Page 6 of 8

Table 3 Correlations between Osteoprotegerin (OPG) and Table 5 Correlation between carotid artery intima media
different parameters in the patients thickness and different parameters of the patients
Parameters Osteoprotegerin (OPG) Parameters Rt. CAIMT Lt CAIMT
r p-value r p r p
Age - 0.082 0.649 Age +0.172 0.340 +0.083 0.646
Duration of disease (years) - 0.063 0.729 Disease duration (years) +0.195 O.276 +0.125 0.487
Body mass index (BMI) +0.331 0.060 BMI +0.123 0.494 +0.143 0.427
Serum ferritin - 0.086 0.633 Serum ferritin +0.200 0.264 +0.059 0.744
Serum cholesterol - 0.205 0.253 Serum cholesterol −0.086 0.633 −0.087 0.631
High density lipoproteins (HDL-C) - 0.263 0.140 TG +0.275 0.121 +0.453 0.008
a
Low density lipoproteins (LDL-C) - 0.318 0.071 HDL-C +0.164 0.361 +0.048 0.790
Triglycerides (TG) +0.374 0.032 LDL-C −0.235 0.188 −0.225 0.207
Atherogenic index of plasma (AIP) +0.263 0.140 AIP +0.155 0.388 +0.446 0.009
C-reactive protein (CRP) - 0.173 0.336 CRP −0.115 0.525 −0.129 0.473
Right carotid artery intima media +0.549 0.001 OPG +0.411 0.018 +0.390 0.025
thickness (Rt. CAIMT)
SGOT +0.387 0.026 +0.003 0.985
Left carotid artery intima media +0.479 0.001
SGPT +0.307 0.082 +0.022 0.903
thickness (Lt. CAIMT)
CAMIT carotid artery intima media thickness, BMI Body mass index, r
Aspartate transaminase (AST) - 0.170 0.344 Spearman’s rank correleation coefficient, TG Triglycerides, p < 0.05 is significant,
Alanine transaminase (ALT) - 0.294 0.097 AIP Atherogenic index of plasma, OPG osteoprotegerin, SGOT Aspartate
transaminase, SGPT Alanine transaminase, CRP C-reactive protein, HDL-C High
r correlation coefficient density lipoproteins cholesterol, LDL-C Low density lipoprote
a
Pearson’s correleation coefficient
p < 0.05 is significant
as risk factor of hypertriglyceridemia due to its negative
controls, or significant correlations with CAIMT. However, impact on extra-hepatic lipolytic activity [26].
results of the hematologic and biochemical investigations of Significantly high S. ferritin was found among B-TM pa-
our B-TM patients displayed very peculiar metabolic model tients in our series, this finding was in agreement with
with; significant anemia, sky high increase in serum ferritin, many previous researchers [27, 28]. Poor compliance to
and dyslipidemia which presented as high triglyceride and iron chelation was obvious among our patient (51%), rep-
atherogenic index of plasma (AIP) but associated with low resented an important and probably correctable cause of
total cholesterol, LDL-C and HDL-C in patients as com- iron overload. High iron burden may increase patient’s
pared with comparisons. risk for atherosclerosis by excess free radicle production
Hemolytic anemia might be a predisposing factor for [29, 30]. Moreover, Mansi and Aburjai documented posi-
atherosclerosis by several mechanisms. First, erythrocyte tive correlation between S. ferritin and triglyceride level
release arginase enzyme during hemolysis coupled with “an important predictor of atherosclerosis” [31]. Against
the liberation of cell –free hemoglobin [23] contribute to our expectation, no significant correlation could be de-
dysregulated arginine metabolism with low arginine/orni- tected between CAIMT and S. ferritin, and similar result
thine ratio. These metabolic derangements limit the avail- was previously documented by Tantawy and his colleague
ability of arginine to nitric oxide synthase and lead to [21]. This data suggested that non-transferrin bound iron
vascular dysfunction by disturbing the bioavailability of accumulation at cellular level with subsequent macro-
nitric oxide (NO) [24, 25]. Second, anemia was described phage activation may be the triggering for development of
atherosclerosis rather than high serum ferritin level [32].
Table 4 Radiological parameters of the studied group Children with beta thalassemia are at increased risk of de-
CAMIT (mm) Patients (N = 65) Controls (N = 50) MW p-value veloping premature atherosclerosis because of dyslipidemia
Right carotid artery (IMT)
[21]. Lipid profiles have been described by several investiga-
tors but with conflicting results [26, 33–36]. Most of them
Mean ± SD 0.62 ± 0.20 0.29 ± 0.07 5.142 <0.001
including our team have shared common findings of low-
Range 0.4 – 1.1 0.2 – 0.4 ered total cholesterol, LDL-C, HDL-C [27, 34–39]. Values
Left carotid artery (IMT) from our B-TM patients and many other researchers showed
Mean ± SD 0.66 ± 0.17 0.29 ± 0.05 6.609 <0.001 elevated triglyceride levels (TG) [33, 34, 36–38], while others
Range 0.4 – 1.2 0.2 – 0.4 described TG levels as being not- significantly different from
MW Mann Whitney U test, CAMIT Carotid artery intima media thickness,
controls [35, 39]. CAIMT in the present study was positively
p < 0.05 is significant, mm millimeter correlated with S. TG and atherogenic index of plasma.
Sherief et al. BMC Pediatrics (2017) 17:69
Dyslipidemia in thalassemia patients has been previously
explained by several researchers who speculated different
pathophysiologic pathways; plasma dilution because of
anemia, accelerated erythropoiesis with excess cholesterol
uptake by macrophages and histocytes of the reticuloen-
dothelial system, defective liver synthetic function because
of iron overload, macrophage activation with cytokine re-
lease and hormonal disturbance [33, 40, 41] while reduced
extra-hepatic lipolytic activity might be responsible for the
increased TG in B-TM patients [33].
It was surprising that our B-TM patients had early ath-
erosclerosis despite low LDL-C, this observation is pos-
sibly due to oxidative change of LDL-C to “atherogenic
LDL” by unbalanced oxidant- antioxidant milieu in thal-
assemia patients [42–45].
Atherogenic index of plasma (AIP) is a marker of
atherogenicity since it is related directly to atheroscler-
osis; it is calculated as log (TG/HDL-C). Hypertriglyc-
eridemia will increase the activity of hepatic lipase which
results in HDL-C degradation with subsequent increased
risk of coronary atherosclerosis [46]. In the present
work, there was significant increase in AIP ratio for B-
TM patients than for the healthy controls and also, it
displayed positive correlation with CAIMT. These find-
ing was in agreement with those by Najajou et al. and
Daniel et al. who reported high predictive value of AIP
for development of atherosclerosis [47, 48].
Rt. CAIMT showed significant correlation with SGOT
(AST) but not with other liver enzyme, these findings might
point to hemolysis in triggering atherosclerosis in our pa-
tients as hemolysis is the main source of elevated AST [49].
In the current study we had the chance to ascertain
that Osteoprotegerin (OPG) was significantly raised in
patients as compared with comparisons (P < 0.001), and
to document the positive correlation that displayed be-
tween OPG, CIMT, and TG. Elevated OPG level was
suggested; as a marker of arterial damage [50], as pre-
dictor of coronary artery diseases, [51] and overall car-
diovascular morbidity and mortality [52], but as far as
our knowledge our team was the first to investigate the
relationship between atherosclerosis as expressed by
CIMT and OPG in B-TM patients.
Results from two large cohort studies for the validity of
OPG assay as a predictor of atherosclerosis and coronary
artery diseases were supporting for its clinical application
in predicting atherosclerosis in asymptomatic high-risk
individuals [53], [54]. Positive correlation between OPG
and CIMT observed among our B-TM patientshighlighted
its importance as promising biomarker of subclinical
atherosclerosis detection.
Conclusions
Premature subclinical atherosclerosis was documented
among Egyptian beta thalassemia major patients by
Page 7 of 8
evaluating their carotid artery intima media thickness
(CAIMT). It was correlated well with dyslipidemia and
serum osteoprotogrin, a finding that highlighted the pos-
sible validity of OPG assay as an early predictor of athero-
sclerosis in thalassemia children. However, multicenter
wide scale research is warranted to evaluate OPG assay cut-
off value, its sensitivity and specificity as reliable biomarker
for diagnosis of atherosclerosis.
Abbreviations
B-TM: Beta-thalassemia major.; CAIMT: Carotid artery intima media thickness;
OPG: Osteoprotegerin
Acknowledgements
Funding
No funds were available to the current work.
Availability of data and materials
available upon request.
Authors’ contributions
LMS, put the conception and design of the study, shared in acquisition, analysis
and interpretation of data and drafting of the manuscript. AA, MA, MN, WM:
shared in acquisition, analysis and interpretation of data. HS, ME, NMK: shared in
drafting of the manuscript and revising it critically for important intellectual
contents. OD was responsible for duplex ultrasound and its interpretation.
OA was responsible for laboratory testing and its interpretation. All authors
have given final approval of the version to be published.
Competing interests
The authors declare that they have no competing interests.
Consent for publication
we have obtained consent to publish from the participant (or legal parent or
guardian for children) to report individual patient data.
Ethics approval and consent to participate
The study protocol was approved by the research and ethical committee of
Faculty of Medicine, Zagazig University and written informed consent was
taken from parents or guardian of each participant.
Author details
1
Pediatrics and Pediatric Hematology and Oncology, Faculty of Medicine,
Zagazig University, Zagazig, Egypt. 2Radiology Department, Zagazig
University, Zagazig, Egypt. 3Pediatrics Department, Zagazig University,
Zagazig, Egypt. 4Pediatric Department, Cairo University, Giza, Egypt. 5Pediatric
Department, Tanta University, Tanta, Egypt. 6Biochemistry Department,
Zagazig University, Zagazig, Egypt.
Received: 23 October 2015 Accepted: 23 February 2017
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